Figure 6

Proliferation response of M14(5–16) with shRXRg infection and western blot of these cells. A – M14(5–16), the SCR infected and the shRXRγ infected subline was grown in 2% fetal bovine serum RPMI in the presence of 1 μmol/L of LGD1069, TTNPB or the combination for 6 days. Cell growth was analyzed using a nonradioactive cell proliferation assay. Proliferation was compared to that of cells grown in volume equivalent vehicle (DMSO – represented by the line). Each treatment condition led to a significant decrease in proliferation compared to control (p < 0.001). The combination of LGD/TTNPB had a statistically significantly greater decrease in proliferation than each alone (*p = 0.02). Proliferation of the SCR infected M14(5–16) was compared to the native cell line to confirm a similar response and then the shRXRγ infected cell line was compared to the SCR condition for an assessment of attenuation of decreased proliferation. Proliferation was significantly attenuated compared to the M14(5–16) SCR subline monotherapy conditions (p ≤ 0.02). Columns, mean; bars, SEM. B – 60 μg of nuclear protein extract from M14(5–16), the SCR shRNA infected control cell and a clone of shRXRg infected cells were size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ and RXRα primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. RXRγ is represented as a doublet because of cross-reaction of the RXRγ₁ and RXRγ₂ isoforms. PARP was measured as a loading control.